Multiplex ligation-dependent probe amplification (MLPA)
MLPA was conducted according to the supplied manufacturer’s specifications using the probemix P022-PLP1 (MRC-holland, Amsterdam, Holland). PCR products were then mixed with formamide (HiDi Formamide, Applied Biosystems, Foster City, CA) and fluorescent Genescan 500 LIZ size standard (Applied Biosystems) prior to analysis using an Applied Biosystems ABI 3130xl capillary sequencer (Applied Biosystems). Data collection and export used GeneMapper software (Applied Biosystems). Peak areas of each fragment were compared to those of a control sample to calculate the gene dosage of each amplicon, including those corresponding to each exon of the PLP1 gene.
X chromosome inactivation pattern
The analysis of the X inactivation pattern was based on extend human androgen receptor (HUMARA) assay proposed Bertelsen et al. [8]. Briefly, 200 ng of leukocyte DNA was digested with HpaII (New England Biolabs, Ipswich, MA) at 37 °C for 12 h. PCR amplification of four loci (AR, PCSK1N, SLITRK4, and ZDHHC15) was performed on undigested DNA and HpaII digested DNA. PCR products were subsequently analyzed on an ABI 3130xl Genetic Analyzer (Applied Biosystems) using GeneMapper software (Applied Biosystems). The X-inactivation ratios were calculated as previously described and skewed X chromosome inactivation was considered if this ratio decrease below 20 % in the investigated blood sample [8].
PLP1 gene expression analysis in patient fibroblasts
Total RNA was isolated from fibroblasts of eight controls without copy number variation of the PLP1 gene, of one positive control with a duplication of PLP1, and of our patient using the guanidinium thiocyanate-phenol-chloroform method. Ten microgram was used to generate cDNA using Superscript II first-strand cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA) with oligo (dT) primers, according to the manufacturer’s protocol. Our patient and control cDNAs were used for PLP1 gene expression analysis performed by real-time PCR according to previously reported conditions [9]. Specifically designed primers were used to determined total PLP1 gene expression after normalization to the beta-glucuronidase gene (GUSB) expression using the ΔΔCt method.
Microarray
Creation of a custom array CGH design was performed using the Agilent software eArray (https://earray.chem.agilent.com/earray/), referred to the GRCh37/hg19 assembly to cover the PLP1 genomic region with maximum resolution. The microarray contained a total of 30,032 probes spanning a 20 Mb region surrounding the PLP1 gene (Agilent Technologies, Santa Clara, CA). Those 30,032 probes were distributed between three groups with various probe densities. The first group includes 15,032 probes encompassing 2 Mb (chrX: 102,000,000–104,000,000; corresponding to a 150 bp average probe spacing) around the PLP1 gene and includes most of the PLP1 duplications described so far [10, 11]. A second, largest interval contained 10,000 probes surrounding 8 Mb (chrX: 98,000,000–102,000,000 and chrX: 104,000,000–106,000,000; 800 bp average probe spacing). The last interval was designed to encompass the largest PLP1 rearrangement and includes 5,000 probes spanning 10 Mb (chrX: 93,000,000–98,000,000 and chrX: 106,000,000–113,000,000; 2000 bp average probe spacing) [6].
A HumanCytoSNP-12 (V2.1) pangenomic microarray (Illumina, San Diego, CA USA) was also performed.
Fluorescence in situ hybridization (FISH)
Fluorescence in situ hybridization (FISH) was performed on lymphocytes metaphase spreads according to standard protocols. FISH analysis was carried out with the probe RP11-832L2 spanning 183 Kb (chrX:102,902,650–103,085,315), which encompasses the entire PLP1 gene, together with a subtelomeric probe of the short arm of chromosome 1 (CEB108/T7).
Genome walker
The Universal Genome Walking Kit (Clontech, Palo Alto, CA, USA) was used to identify precise breakpoints of the inserted PLP1-containing X chromosome segment into 1p36. According to the manufacturer’s protocol, the genomic DNA of the patient was completely digested with four separate restriction enzymes (i.e. EcoRV, DraI, PvuII, and SspI) and subsequently ligated to the GenomeWalker adaptors. The GenomeWalker libraries produced were then used as a template for primary PCR amplification. The primers used were the outer adaptor primer provided in the kit and a Xq22 specific primer localized at the boundaries of the duplicated PLP1 segment identified with our highly resolutive PLP1-custom array CGH. A secondary nested PCR using the nested adaptor primer and a nested Xq22 specific primer was then performed. Sequencing of the PCR products was finally achieved if a single or two (considering that both, the normal Xq22 and the 1p36 derivative, alleles could be digested and ligated to the adaptor) specific and major PCR products were visible after electrophoresis through an agarose gel stained with GelRed (Biotium, Hayward, CA, USA). Sequencing was conducted using the nested primers with the BigDye terminator sequencing kit (Applied Biosystems).